This application is a 371 of PCT/EP99/02344 filed Apr. 7, 1999.
The present invention relates to novel dihydropyrimidine compounds, to processes for their preparation and to their use as medicaments, in particular for the treatment and prophylaxis of hepatitis B.
Dihydropyrimidines having cardiovascular action are already known from the publication EP 103 796 A2.
The present invention now provides novel dihydropyrimidine compounds of the general formula (I) 
or their isomeric form (Ia) 
in which
R1 represents phenyl, furyl, thienyl, triazolyl, pyridyl, cycloalkyl having 3 to 6 carbon atoms or represents radicals of the formulae 
xe2x80x83where the abovementioned ring systems are optionally mono- or polysubstituted by identical or different substituents selected from the group consisting of halogen, trifluoromethyl, nitro, cyano, trifluoromethoxy, carboxyl, hydroxyl, (C1-C6)-alkoxy, (C1-C6)-alkoxycarbonyl and (C1-C6)-alkyl, which for its part may be substituted by aryl having 6 to 10 carbon atoms or halogen, and/or the ring systems mentioned are optionally substituted by groups of the formulae xe2x80x94Sxe2x80x94R6, NR7R8, COxe2x80x94NR9R10, SO2xe2x80x94CF3, and xe2x80x94Axe2x80x94CH2xe2x80x94R11, in which
R6 represents phenyl which is optionally substituted by halogen,
R7, R8, R9 and R10 are identical or different, and each represents hydrogen, phenyl, hydroxyl-substituted phenyl, hydroxyl, (C1-C6)-acyl or (C1-C6)-alkyl, which for its part may be substituted by hydroxyl, (C1-C6)-alkoxycarbonyl, phenyl or hydroxyl-substituted phenyl,
A represents a radical O, S, SO or SO2,
R11 represents phenyl which is optionally mono- to polysubstituted by identical or different substituents selected from the group consisting of halogen, nitro, trifluoromethyl, (C1-C6)-alkyl and (C1-C6)-alkoxy,
R2 represents a radical of the formula xe2x80x94XR12 or xe2x80x94NR13R14, in which
X represents a bond or oxygen,
R12 represents hydrogen, straight-chain or branched (C1-C6)-alkoxycarbonyl or a straight-chain, branched or cyclic saturated or unsaturated (C1-C8)-hydrocarbon radical which optionally contains one or two identical or different hetero chain members from the group consisting of O, CO, NH, xe2x80x94NHxe2x80x94(C1-C4)-alkyl, xe2x80x94Nxe2x80x94((C1-C4)-alkyl)2, S and SO2 and which is optionally substituted by halogen, nitro, cyano, hydroxyl, aryl having 6 to 10 carbon atoms or aralkyl having 6 to 10 carbon atoms, heteroaryl or a group of the formula xe2x80x94NR15R16, in which
R15 and R16 are identical or different, and each represents hydrogen, benzyl or (C1-C6)-alkyl,
R13 and R14 are identical or different, and each represents hydrogen, (C1-C6)-alkyl or cycloalkyl having 3 to 6 carbon atoms,
R3 represents hydrogen, amino or represents a radical of the formula 
xe2x80x83or
represents formyl, cyano, trifluoromethyl or pyridyl, or
represents a straight-chain, branched or cyclic saturated or unsaturated hydrocarbon radical having up to 8 carbon atoms which is optionally mono- or polysubstituted by identical or different substituents from the group consisting of aryloxy having 6 to 10 carbon atoms, azido, cyano, hydroxyl, carboxyl, (C1-C6)-alkoxycarbonyl, a 5- to 7-membered heterocyclic ring, (C1-C6)-alkylthio and (C1-C6)-alkoxy, which for its part may be substituted by azido or amino, and/or is substituted by triazolyl, which for its part may be substituted up to 3 times by (C1-C6)-alkoxycarbonyl, and/or may be substituted by groups of the formulae xe2x80x94OSO2xe2x80x94CH3 or (CO)axe2x80x94NR17R18, in which
a represents a number 0 or 1,
R17 and R18 are identical or different, and each represents hydrogen or aryl, aralkyl having 6 to 10 carbon atoms, or represents (C1-C6)-alkyl which is optionally substituted by (C1-C6)-alkoxycarbonyl, hydroxyl, phenyl or benzyl, where phenyl or benzyl are optionally mono- or polysubstituted by identical or different substituents from the group consisting of hydroxyl, carboxyl, (C1-C6)-alkyl and (C1-C6)-alkoxy, or (C1-C6)-alkyl is optionally substituted by groups of the formulae NHxe2x80x94COxe2x80x94CH3 or NHxe2x80x94COxe2x80x94CF3, or
R17 and R18 together with the nitrogen atom form a morpholine, piperidinyl or pyrrolidinyl ring, or
R3 represents phenyl which is optionally substituted by methoxy, or
R2 and R3 together form a radical of the formula 
R4 represents hydrogen, (C1-C4)-alkyl, (C2-C4)-alkenyl, benzoyl or represents acyl having 2 to 6 carbon atoms,
R5 represents pyridyl which is substituted up to 3 times by identical or different substituents from the group consisting of halogen, hydroxyl, cyano, trifluoromethyl, (C1-C6)-alkoxy, (C1-C6)-alkyl, (C1-C6)-alkylthio, carbalkoxy, (C1-C6)-acyloxy, amino, nitro, mono- and (C1-C6)-dialkylamino,
and salts thereof.
In the context of the invention, cycloalkyl having 3 to 6 carbon atoms or (C3-C6)-cycloalkyl represents cyclopropyl, cyclopentyl, cyclobutyl or cyclohexyl. Preference is given to cyclopentyl or cyclohexyl.
Aryl generally represents an aromatic radical having 6 to 10 carbon atoms. Preferred aryl radicals are phenyl and naphthyl.
In the context of the invention, (C1-C6)-acyl represents a straight-chain or branched acyl radical having 1 to 6 carbon atoms. Preference is given to a straight-chain or branched acyl radical having 1 to 4 carbon atoms. Particularly preferred acyl radicals are acetyl and propionyl.
In the context of the invention, (C1-C6)-alkyl represents a straight-chain or branched alkyl radical having 1 to 6 carbon atoms. Examples which may be mentioned are: methyl, ethyl, propyl, isopropyl, tert-butyl, n-pentyl and n-hexyl.
Preference is given to a straight-chain or branched alkyl radical having 1 to 4 carbon atoms.
In the context of the invention, (C2-C6)-alkenyl represents a straight-chain or branched alkenyl radical having 2 to 5 carbon atoms. Preference is given to a straight-chain or branched alkenyl radical having 3 to 5 carbon atoms. Examples which may be mentioned are: ethenyl, propenyl, alkyl, n-pentenyl and n-hexenyl.
In the context of the invention, (C1-C6)-alkoxy represents a straight-chain or branched alkoxy radical having 1 to 6 carbon atoms. Preference is given to a straight-chain or branched alkoxy radical having 1 to 4 carbon atoms. Examples which may be mentioned are: methoxy, ethoxy and propoxy.
In the context of the invention, (C1-C6)-alkylthio represents a straight-chain or branched alkylthio radical having 1 to 6 carbon atoms. Preference is given to a straight-chain or branched alkylthio radical having 1 to 4 carbon atoms. Examples which may be mentioned are: methylthio, ethylthio and propylthio.
In the context of the invention, (C1-C6)-alkoxycarbonyl represents a straight-chain or branched alkoxycarbonyl radical having 1 to 6 carbon atoms. Preference is given to a straight-chain or branched alkoxycarbonyl radical having 1 to 4 carbon atoms. Examples which may be mentioned are: methoxycarbonyl, ethoxycarbonyl and propoxycarbonyl.
A straight-chain, branched or cyclic, saturated or unsaturated (C1-C8)-hydrocarbon radical includes, for example, the above-described (C1-C6)-alkyl, (C2-C6)-alkenyl or (C3-C6)-cycloalkyl, preferably (C1-C6)-alkyl.
The compounds according to the invention may exist in stereoisomeric forms which are related either as image and mirror image (enantiomers), or which are not related as image and mirror image (diastereomers). The invention relates both to the enantiomers or diastereomers and to their respective mixtures. The racemic forms can, just like the diastereomers, be separated in a known manner into the stereoisomerically pure constituents.
The compounds of the present invention include the isomers of the general formulae (I) and (Ia) and mixtures thereof. If R4 is hydrogen, the isomers (I) and (Ia) exist in a tautomeric equilibrium: 
The substances according to the invention may also be present as salts. In the context of the invention, preference is given to physiologically acceptable salts.
Physiologically acceptable salts can be salts of the compounds according to the invention with inorganic or organic acids. Preference is given to salts with inorganic acids, such as, for example, hydrochloric acid, hydrobromoic acid, phosphoric acid or sulphuric acid, or to salts with organic carboxylic or sulphonic acids, such as, for example, acetic acid, maleic acid, fumaric acid, malic acid, citric acid, tartaric acid, lactic acid, benzoic acid, or methanesulphonic acid, ethanesulphonic acid, phenylsulphonic acid, toluenesulphonic acid or naphthalenedisulphonic acid.
Physiologically acceptable salts can also be metal or ammonium salts of the compounds according to the invention. Particular preference is given to, for example, sodium, potassium, magnesium or calcium salts, and also to ammonium salts which are derived from ammonia, or organic amines, such as, for example, ethylamine, di- or triethylamine, di- or triethanolamine, dicyclohexylamine, dimethylarninoethanol, arginine, lysine, ethylenediamine or 2-phenylethylamine.
Preference is given to compounds of the general formulae (I) and (Ia) according to the invention in which
R1 represents phenyl, furyl, thienyl, pyridyl, cyclopentyl or cyclohexyl or represents radicals of the formulae 
xe2x80x83where the abovementioned ring systems are optionally mono- or disubstituted by identical or different substituents selected from the group consisting of halogen, trifluoromethyl, nitro, SO2xe2x80x94CF3, methyl, cyano, trifluoromethoxy, amino, hydroxyl, carboxyl, methoxycarbonyl and radicals of the formulae xe2x80x94COxe2x80x94NHxe2x80x94CH2xe2x80x94C(CH3)3, xe2x80x94COxe2x80x94NH(CH2)2OH, xe2x80x94COxe2x80x94NHxe2x80x94CH2xe2x80x94C6H5, xe2x80x94COxe2x80x94NHxe2x80x94C6H5, xe2x80x94COxe2x80x94NHxe2x80x94(pOH)xe2x80x94C6H4, xe2x80x94Oxe2x80x94CH2xe2x80x94C6H5 or xe2x80x94Sxe2x80x94pClxe2x80x94C6H4,
R2 represents a radical of the formula xe2x80x94XR12 or xe2x80x94NR13R14, in which
X represents a bond or an oxygen atom,
R12 represents hydrogen, (C1-C4)-alkenyl, (C1-C4)-alkoxycarbonyl or (C1-C4)-alkyl which are optionally substituted by pyridyl, cyano, phenoxy, benzyl or by a radical of the formula xe2x80x94NR15R16, in which
R13 and R16 are identical or different, and each represents hydrogen, benzyl or (C1-C4)-alkyl,
R13 and R14 are identical or different, and each represents hydrogen, (C1-C4)-alkyl or cyclopropyl,
R3 represents hydrogen, amino or a radical of the formula 
xe2x80x83or represents formyl, cyano, trifluoromethyl, cyclopropyl or pyridyl, or represents (C1-C4)-alkyl which is optionally substituted by halogen, (C1-C4)-alkoxycarbonyl, hydroxyl or by triazolyl, which for its part may be substituted up to 3 times by (C1-C4)-alkoxycarbonyl, and/or alkyl is optionally substituted by groups of the formulae xe2x80x94OSO2xe2x80x94CH3 or (CO)axe2x80x94NR17R18, in which
a represents a number 0 or 1,
R17 and R18 are identical or different, and each represents hydrogen, phenyl or benzyl, or represents C1-C4-alkyl which is optionally substituted by (C1-C4)-alkoxycarbonyl, hydroxyl, phenyl or benzyl, where phenyl or benzyl are optionally mono- or polysubstituted by identical or different substituents from the group consisting of hydroxyl, carboxyl, (C1-C4)-alkyl and (C1-C4)-alkoxy, and/or (C1-C4)-alkyl is optionally substituted by radicals of the formulae xe2x80x94NHxe2x80x94COxe2x80x94CH3 or xe2x80x94NHxe2x80x94COxe2x80x94CF3, or
R17 and R18 together with the nitrogen atom form a morpholine, piperidinyl or pyrrolidinyl ring, or
R3 represents phenyl which is optionally substituted by methoxy, or
R2 and R3 together form a radical of the formula 
R4 represents hydrogen, methyl, benzoyl or acetyl,
R5 represents pyridyl which is substituted up to 2 times by identical or different substituents from the group consisting of fluorine, chlorine, bromoine, (C1-C4)-alkoxy and (C1-C4)-alkyl,
and salts thereof.
Particular preference is given to compounds of the general formulae (I) and (Ia) according to the invention, in which
R1 represents phenyl, furyl, thienyl, pyridyl, cyclopentyl, cyclohexyl or represents radicals of the formulae 
xe2x80x83where the abovementioned ring systems are optionally substituted up to 2 times by identical or different substituents selected from the group consisting of fluorine, chlorine, bromine, iodine, hydroxyl, trifluoromethyl, nitro, SO2xe2x80x94CF3, methyl, cyano, amino, trifluoromethoxy, carboxyl, methoxycarbonyl and radicals of the formulae xe2x80x94COxe2x80x94NHxe2x80x94CH2xe2x80x94C(CH3)3, xe2x80x94COxe2x80x94NH(CH2)2OH, xe2x80x94COxe2x80x94NHxe2x80x94CH2-C6H5, xe2x80x94COxe2x80x94NHxe2x80x94C6H5, xe2x80x94COxe2x80x94NHxe2x80x94(pOH)xe2x80x94C6H4, xe2x80x94Oxe2x80x94CH2xe2x80x94C6H5 or xe2x80x94Sxe2x80x94pClxe2x80x94C6H4,
R2 represents a radical of the formula xe2x80x94XR12 or xe2x80x94NR13R14, in which
X represents a bond or an oxygen atom,
R12 represents hydrogen, (C1-C3)-alkenyl, (C1xe2x80x94C,)-alkoxycarbonyl or (C1-C4)-alkyl which are optionally substituted by pyridyl, cyano, phenoxy, benzyl or by a radical of the formula xe2x80x94NR15R16, in which
R15 and R16 are identical or different, and each represents hydrogen or methyl,
R13 and R14 are identical or different, and each represents hydrogen, (C1-C3)-alkyl or cyclopropyl,
R3 represents hydrogen, amino or represents a radical of the formula 
xe2x80x83represents formyl, cyano, trifluoromethyl, cyclopropyl or pyridyl, or
represents (C1-C,)-alkyl which is optionally substituted by fluorine, chlorine, (C1-C3)-alkoxycarbonyl, hydroxyl or by triazolyl, which for its part may be substituted up to 3 times by (C1-C3)-alkoxycarbonyl, and/or alkyl is optionally substituted by groups of the formulae xe2x80x94OSO2xe2x80x94CH3 or (CO)axe2x80x94NR17R18, in which
a represents a number 0 or 1,
R17 and R18 are identical or different, and each represents hydrogen, phenyl or benzyl, or represents (C1-C3)-alkyl which is optionally substituted by (C1-C3)-alkoxycarbonyl, hydroxyl, phenyl or benzyl, where phenyl or benzyl are optionally mono- or disubstituted by identical or different substituents from the group consisting of hydroxyl, carboxyl, (C1-C3)-alkyl and (C1-C3)-alkoxy, and/or (C1-C4)-alkyl is optionally substituted by radicals of the formulae xe2x80x94NHxe2x80x94COxe2x80x94CH3 or xe2x80x94NHxe2x80x94COxe2x80x94CF3, or
R17 and R18 together with the nitrogen atom form a morpholine, piperidinyl or pyrrolidinyl ring, or
R3 represents phenyl which is optionally substituted by methoxy, or
R2 and R 3together form a radical of the formula 
R4 represents hydrogen, methyl, benzoyl or acetyl,
R5 represents pyridyl which is substituted up to 2 times by identical or different substituents from the group consisting of fluorine, chlorine, (C1-C3)-alkoxy and (C1-C3)-alkyl,
and salts thereof.
Very particular preference is given to compounds of the general formulae (I) and (Ia) according to the invention, in which
R1 represents phenyl which is optionally substituted up to 2 times by identical or different substituents from the group consisting of fluorine, chlorine, bromine, iodine, methyl and nitro,
R2 represents xe2x80x94XR12 in which X represents oxygen and R12 represents straight-chain or branched alkyl having up to 4 carbon atoms,
R3 represents methyl, ethyl or cyclopropyl, or
R2 and R3 together form a radical of the formula 
R4 represents hydrogen or acetyl, and
R5 represents pyridyl which is substituted up to two times by identical or different substituents from the group consisting of fluorine and chlorine,
and salts thereof.
Even more preference is given to compounds of the general formula (I) or (Ia) according to the invention in which R5 represents 2-pyridyl which can be substituted by 1 to 2 fluorine atoms.
Very particular preference is also given to those compounds of the general formulae (I) and (Ia) according to the invention which are listed in Table A:
Very particular preference is given to the following compounds: 
The compounds of the general formula (I) according to the invention can be prepared by
[A] reacting aldehydes of the general formula (II)
R1xe2x80x94CHOxe2x80x83xe2x80x83(II)
in which
R1 is as defined above,
with amidines or their hydrochlorides of the formula (III) 
in which
R5 is as defined above,
and compounds of the general formula (IV)
xe2x80x83R3xe2x80x94COxe2x80x94CH2xe2x80x94COxe2x80x94R2xe2x80x83xe2x80x83(IV)
in which
R2 and R3 are each as defined above,
if appropriate in the presence of inert organic solvents, with or without addition of base or acid, or
[B] reacting compounds of the general formula (V) 
in which
R1, R2 and R3 are each as defined above,
with amidines of the general formula (III) 
in which
R5 is as defined above,
if appropriate in the presence of inert organic solvents at temperatures between 20xc2x0 C. and 150xc2x0 C., with or without addition of base or acid, or
[C] reacting aldehydes of the general formula (II)
R1xe2x80x94CHOxe2x80x83xe2x80x83(II)
in which
R1 is as defined above,
with compounds of the general formula (VI) 
in which
R2 and R3 are each as defined above,
and amidines of the general formula (III) as described above, or
[D] reacting aldehydes of the general formula (II) with compounds of the general formula (IV) and imino ethers of the general formula (VII) 
in which
R5 is as defined above, and
R1 represents (C1-C4)-alkyl,
in the presence of ammonium salts.
The processes according to the invention can be illustrated using the following schemes as examples:
[A] 
Solvents which are suitable for all process variants A, B, C and D are all inert organic solvents. These preferably include alcohols, such as ethanol, methanol, isopropanol, ethers, such as dioxane, diethyl ether, tetrahydrofuran, glycol monomethyl ether, glycol dimethyl ether or glacial acetic acid, dimethyl formamide, dimethyl sulphoxide, acetonitrile, pyridine and hexamethylphosphoric triamide.
The reaction temperatures can be varied within a relatively wide range. In general, the reaction is carried out between 20 and 150xc2x0 C., but preferably at the boiling point of the solvent in question.
The reaction can be carried out at atmospheric pressure, or else at elevated pressure. In general, the reaction is carried out at atmospheric pressure.
The reaction can be carried out with or without addition of base or acid; however, it has been found that a reaction according to the invention is preferably carried out in the presence of relatively weak acids, such as, for example, acetic acid or formic acid.
The aldehydes of the general formula (II) used as starting materials are known or can be prepared by methods known from the literature (cf. T. D. Harris and G. P. Roth, J. Org. Chem. 44, 146 (1979), German Offenlegungsschrift 2 165 260, July 1972, German Offenlegungsschrift 2 401 665, July 1974, Mijano et al., Chem. Abstr. 59, (1963), 13 929 c, E. Adler and H.-D. Becker, Chem. Scand. 15, 849 (1961), E. P. Papadopoulos, M. Mardin and Ch. Issidoridis, J. Org. Chem. Soc. 78, 2543 (1956)).
The ylidene-xcex2-keto esters of the formula (V) used as starting materials can be prepared by methods known from the literature [cf. G. Jones, xe2x80x9cThe Knoevenagel Condensationxe2x80x9d, in Organic Reactions, Vol. XV, 204 ff. (1967)].
The enaminocarboxylic esters of the formula (VI) and the imino ethers of the general formula (VII) used as starting materials are known or can be prepared by methods known from the literature [cf. S. A. Glickman and A. C. Cope, J. Am. Chem. Soc. 67, 1017 (1945)].
The xcex2-ketocarboxylic esters of the general formula (IV) used as starting materials are known or can be prepared by methods known from the literature [for example D. Borrmann, xe2x80x9cUmsetzung von Diketen mit Alkoholen, Phenolen und Mercaptanenxe2x80x9d, in Houben-Weyl, Methoden der organischen Chemie, Vol. VII/4, 230 ff (1968); Y. Oikawa, K. Sugano and O. Yonemitsu, J. Org. Chem. 43, 2087 (1978)].
Some of the compounds of the general formula (III) are known, or, in the case where R5 is difluorinated pyridyl, are novel, and they can be prepared by reacting compounds of the formula (VIII)
R5xe2x80x94CNxe2x80x83xe2x80x83(VIII)
in which
R5 is as defined above,
in the customary way via the imino ethers and finally with ammonium chloride in methanol [cf. in this context W. K. Fife, Heterocycles 22, 93-96 (1984); T. Sakamoto, S. Kaneda, S. Nishimura, H. Yamanaka, Chem. Pharm. Bull. 33, 565-571 (1986)] or other processes known from the literature, such as, for example, Garigipati, Tetrahedron Lett. 1990, pp. 1969-1972, Boere et al., J. Organomet. Chem. 1987, 331, 161, Caton et al., J. Chem. Soc. 1967, 1204.
All process steps are carried out at atmospheric pressure and in a temperature range of from 0xc2x0 C. to 130xc2x0 C., preferably from 20xc2x0 C. to 100xc2x0 C.
Thus, the invention also relates to an intermediate of the formula below 
and its salts from which preferred end products can be prepared. With respect to the salts of this compound, reference is made to the abovementioned acid addition salts and in particular to the hydrochloride. This compound is prepared as described in the examples, and, in this respect, reference is also made to the reaction scheme shown below.
The compounds of the formula (VIII) are known per se or can be prepared by known processes similarly to Example I and II by reacting pyridines of the general formula (IX)
R5xe2x80x94Hxe2x80x83xe2x80x83(IX)
in which the hydrogen is ortho to the nitrogen and in which R5 is as defined above, initially at from 50 to 150xc2x0 C., preferably at 100xc2x0 C., in H2O2/glacial acetic acid to give the corresponding N-oxides, followed by a reaction with trimethylsilyl cyanide (TMSCN) by processes known from the literature in the abovementioned inert solvents, preferably acetonitrile, THF, toluene at room temperature or at reflux temperature, if appropriate with addition of bases such as triethylamine or DBU,
or by replacing, in compounds of the formula (X) 
in which Y and Z represent the substitution radicals of the pyridyl ring mentioned under R5, the chlorine with cyanide, using cyanides, such as potassium cyanide or copper cyanide,
or by reacting, in the case where R5 represents difluoropyridyl, compounds of the formula (XI) 
in which Yxe2x80x2 and Zxe2x80x2 are identical or different, and each represents chlorine or bromine, with alkali metal or ammonium fluorides, preferably potassium fluoride, by processes known from the literature, in polar solvents, such as, for example, polyglycols and ethers thereof, DMSO or sulpholane, if appropriate with addition of phase-transfer catalysts, in a halogen-fluorine exchange reaction.
Thus, the invention also relates to a compound of the formula below from which the corresponding amidine intermediate can be prepared in the manner described in the examples: 
The above process is, with respect to the 3,5-difluoropyridyl compounds, illustrated in an exemplary manner by the following reaction scheme: 
The antiviral activity of the compounds according to the invention was investigated following the methods described by Sells et al. (M. A. Sells, M.-L. Chen, and G. Acs (1987) Proc. Natl. Acad. Sci. 84, 1005-1009) and Korba et al. (B. E. Korba and J. L. Gerin (1992) Antiviral Research 19, 55-70).
The antiviral tests were carried out in 96-well microtitre plates. Only growth medium and HepG2.2.15 cells were added to the first vertical row of the plate. This row served as virus control.
Stock solutions of the test compounds (50 mM) were initially dissolved in DMSO, and further dilutions were prepared in the growth medium of HepG2.2.15. The compounds according to the invention, usually in a test concentration of 100 xcexcM (1st test concentration), were in each case pipetted into the second vertical test row of the microtitre plate and subsequently diluted, by a factor of 2 each time, up to 210-fold, using growth medium plus 2% of foetal calf serum (volume 25 xcexcl).
225 xcexcl of a HepG2.2.15 cell suspension (5xc3x97104cells/ml) in growth medium plus 2% foetal calf serum were then added to each well of the microtitre plate.
The test batch was incubated at 37xc2x0 C., 5% CO2, for 4 days.
The supernatant was subsequently siphoned off and discarded, and 225 xcexcl of freshly prepared growth medium were added to the wells. Once more, the compounds according to the invention were added, in each case as a solution 10-fold-concentrated, in a volume of 25 xcexcl. The batches were incubated for another 4 days.
Before the supernatants were harvested for determining the antiviral effect, the HepG2.2.15 cells were examined under the light microscope or by biochemical detection methods (for example Alamar Blue staining or Trypan Blue staining) for cytotoxic changes.
The supernatants were subsequently harvested and, by means of reduced pressure, siphoned onto 96-well dot blot chambers covered with a nylon membane (in accordance with the specifications of the manufacturer).
Substance-induced cytotoxic or cytostatic changes in the HepG2.2.15 cells were determined as changes in the cell morphology, for example under a light microscope. Such substance-induced changes of the HepG2.2.15 cells in comparison with untreated cells could be observed, for example, as cell lysis, vacuolization or changed cell morphology. 50% cytotoxicity (Tox.-50) means that 50% of the cells have a morphology which is similar to the corresponding cell control.
The compatibility of some of the compounds according to the invention was additionally tested on other host cells, such as, for example, HeLa cells, primary peripheral human blood cells or transformed cell lines, such as H-9 cells. At concentrations of the compounds according to the invention of  greater than 10 xcexcM, no cytotoxic changes were observed.
After transfer of the supernatants onto the nylon membrane of the blot apparatus (see above), the supernatants of the HepG2.2.15 cells were denatured (1.5 M NaCl/0.5 N NaOH), neutralized (3 M NaCl/0.5 M Tris HCl, pH 7.5) and washed (2xc3x97SSC). By incubation of the filters at 120xc2x0 C. for 2-4 hours, the DNA was subsequently baked onto the membrane.
The viral DNA of the treated HepG2.2.15 cells on the nylon filters was usually detected using non-radioactive digoxygenin-labelled hepatitis B-specific DNA probes which were in each case in accordance with the specifications of the manufacturer labelled with digoxygenin, purified and used for hybridization.
The prehybridization and hybridization was carried out in 5xc3x97SSC, 1xc3x97blocking reagent, 0.1% N-lauroylsarcosine, 0.02% SDS and 100 xcexcg of DNA from herring sperm. The prehybridization was carried out at 60xc2x0 C. for 30 minutes and the specific hybridization was carried out using 20 to 40 ng/ml of the digoxygenated denatured HBV-specific DNA (14 hours, 60xc2x0 C.). The filters were subsequently washed.
The digoxygenin-labelled DNA was detected immunologically in accordance with the specifications of the manufacturer:
The filters were washed and prehybridized in a blocking agent (in accordance with the specifications of the manufacturer). They were subsequently hybridized for 30 minutes using an anti-DIG antibody linked to alkaline phosphatase. After a washing step, the substrate of alkaline phosphatase, CSPD, was added, incubated with the filters for 5 minutes, subsequently wrapped in plastic film and incubated at 37xc2x0 C. for a further 15 minutes. The chemiluminescence of the Hepatitis B-specific DNA signals was visualized by exposition of the filters on an X-ray film (incubation, depending on the signal strength: 10 minutes to 2 hours).
The half-maximum inhibitory concentration (IC-50, inhibitory concentration 50%) was determined as the concentration at which the hepatitis B-specific band was reduced by 50% in comparison with an untreated sample by the compound according to the invention.
Surprisingly, the treatment of the hepatitis B virus-producing HepG2.2.15 cells with the compounds according to the invention resulted in a reduction of viral DNA in the cell culture supernatant, the viral DNA being released by the cells into the cell culture supernatant in the form of virions.
The compounds according to the invention have a novel unforeseeable and useful action against viruses. Surprisingly, they are antivirally active against hepatitis B (HBV) and are therefore suitable for treating virus-induced diseases, in particular acute and chronically persisting HBV virus infections. A chronic viral disease caused by HBV can lead to clinical pictures of various gravity; as is known, chronic hepatitis B virus infection frequently results in cirrhosis of the liver and/or hepatocellular carcinoma.
Examples which may be mentioned of areas of indication for the compounds usable according to the invention are:
The treatment of acute and chronic virus infections which may lead to an infectious hepatitis, for example infections with hepatitis B viruses. Particular preference is given to the treatment of chronic hepatitis B infections and the treatment of acute hepatitis B virus infection.
The present invention encompasses pharmaceutical formulations which, in addition to non-toxic inert pharmaceutically acceptable excipients, contain one or more compounds of the formulae (I), (Ia) or of Table A or which comprise one or more active compounds of the formulae (I), (Ia) and (Ib), and also encompasses processes for producing these formulations.
In the abovementioned pharmaceutical formulations, the active compounds of the formulae (I), (Ia) and (Ib) should be present in a concentration of approximately 0.1-99.5% by weight, preferably of approximately 0.5-95% by weight of the total mixture.
The abovementioned pharmaceutical formulations may, in addition to the compounds of the formulae (I), (Ia) and (Ib), also contain further pharmaceutically active compounds.
The abovementioned pharmaceutical formulations are produced in a customary manner by known methods, for example by mixing the active compound(s) with the excipient(s).
In general, it has been found to be advantageous both in human and veterinary medicine to administer the active compound(s) in total amounts of from approximately 0.5 to approximately 500, preferably 1-100 mg/kg of body weight per 24 hours, if appropriate in the form of several individual doses, to obtain the desired results. An individual dose preferably contains the active compound(s) in amounts of from approximately 1 to approximately 80, in particular 1-30 mg/kg of body weight. However, it may be necessary to deviate from the specified dosages, depending on the nature and the body weight of the object to be treated, the nature and the severity of the disease, the formulation type and the administration of the medicament, and the time or interval within which administration is carried out.